Therapeutic agent for chronic arthritides diseases of childhood-related diseases

ABSTRACT

A therapeutic agent for chronic arthritides diseases of childhood-related diseases, for example chronic arthritides diseases of childhood, Still&#39;s disease and the like, comprising an interleukin-6 (IL-6) antagonist as an active ingredient.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This Application is a Continuation of application Ser. No. 13/064,953,filed Apr. 28, 2011, which is a Divisional of application Ser. No.11/704,233, filed Feb. 9, 2007, which is a Divisional of applicationSer. No. 10/473,165, which entered the U.S. national phase on Sep. 29,2003, from PCT/JP02/03312 filed Apr. 2, 2002, which claims priority fromJapanese patent applications 2001-109131 filed Apr. 6, 2001 and2001-103627 filed Apr. 2, 2001. The entire contents of theaforementioned applications are incorporated herein by reference

FIELD OF THE INVENTION

The present invention relates to a therapeutic agent for “chronicarthritides diseases of childhood-related diseases” comprising aninterleukin-6 (IL-6) antagonist as an active ingredient. Chronicarthritides diseases of childhood-related diseases include chronicarthritides diseases of childhood, Still's disease and the like.

BACKGROUND ART

IL-6 is a cytokine called B-cell stimulating factor 2 (BSF2) orinterferon β2. IL-6 was discovered as a differentiation factorresponsible for activation of B-lymphatic cells (Hirano, T. et al.,Nature (1986) 324, 73-76). Thereafter, it was found to be amultifunctional cytokine that influences the function of various cells(Akira, S. et al., Adv. in Immunology (1993) 54, 1-78). IL-6 has beenreported to induce the maturation of T lymphatic cells (Lotz, M. et al.,J. Exp. Med. (1988) 167, 1253-1258).

IL-6 propagates its biological activity through two proteins on thecell. One is a ligand-binding protein, IL-6 receptor, with a molecularweight of about 80 kD to which IL-6 binds (Taga T. et al., J. Exp. Med.(1987) 166, 967-981; Yamasaki, K. et al., Science (1987) 241, 825-828).IL-6 receptor exists not only in a membrane-bound form that penetratesand is expressed on the cell membrane but also as a soluble IL-6receptor consisting mainly of the extracellular region.

The other is non-ligand-binding membrane-bound protein gp130 with amolecular weight of about 130 kD that takes part in signal transduction.IL-6 and IL-6 receptor form an IL-6/IL-6 receptor complex, to whichgp130 is bound, and thereby the biological activity of IL-6 ispropagated into the cell (Taga et al., Cell (1989) 58, 573-581).

IL-6 antagonists are substances that inhibit the transduction of IL-6biological activities. Up to now, there have been known antibodies toIL-6 (anti-IL-6 antibodies), antibodies to IL-6 receptor (anti-IL-6receptor antibodies), antibodies to gp130 (anti-gp130 antibodies),reshaped IL-6, IL-6 or IL-6 receptor partial peptides, and the like.

Antibodies to IL-6 receptor have been described in a number of reports(Novick D. et al., Hybridoma (1991) 10, 137-146; Huang, Y. W. et al.,Hybridoma (1993) 12, 621-630; International Patent Application WO95-09873; French Patent Application FR 2694767; United States PatentU.S. Pat. No. 5,216,128). A humanized PM-1 antibody was obtained byimplanting the complementarity determining region (CDR) of a mouseantibody PM-1 (Hirata et al., J. Immunology (1989), one of anti-IL-6receptor antibodies, 143, 2900-2906) into a human antibody(International Patent Application WO 92-19759).

Chronic arthritides diseases of childhood are diseases comprising mainlychronic arthritis that develops at less than 16 years of age and is themost prevalent disease among the collagen diseases that develop inchildren. Unlike rheumatoid arthritis (RA) in adults, they are notconsidered to be a homogeneous disease and have a variety of diseasetypes, and therefore they tend to be dealt with as a disease entitydifferent from rheumatoid arthritis in adults.

As the name of chronic arthritides diseases of childhood, “juvenilerheumatoid arthritis (JRA)” has been used in Japan according to thediagnostic criteria in the United States, whereas in Europe the term“juvenile chronic arthritis (JCA)” is mainly used. Recently, terms suchas idiopathic chronic arthritis (ICA) and juvenile idiopathic arthritis(JIA) have been used.

The disease types of chronic arthritides diseases of childhood have beencategorized in various ways. According to the American College ofRheumatology (ACR), they are divided, as arthritic diseases that developin children less than 16 years old and persist for six weeks or longer,into three disease types: 1) systemic onset JRA, 2) polyarticular, 3)pauciarticular (ARA classification) (JRA Criteria Subcommittee of theDiagnostic and Therapeutic Criteria Committee of the American RheumatismAssociation Arthritis Rheum 20 (Suppl): 195, 1977). In Europe, theEuropean League Against Rheumatism (EULAR) has made a classificationthat states that, though it differs from the above ARA classification inthat the duration of arthritis is three months or more and arthritis dueto psoriasis, ankylosing spondylitis etc. has been excluded, the threedisease types are similar (Bulletin 4, Nomenclature and classificationof Arthritis in Children. Basel, National Zeitung AG, 1977).

Recently, a revision of the classification has been attempted, and theInternational League of Associations for Rheumatology (ILAR) proposed in1995 a classification plan of Idiopathic Arthritides of Childhood (FinkC W, Proposal for the development of classification criteria foridiopathic arthritides of childhood. J. Rheumatol., 22: 1566 (1995)),and in 1997 the revision was proposed as an ILAR plan (Southwood T R,Classifying childhood arthritis, Ann. Rheum. Dis. 56: 79 (1997)). Thisclassification provides division into: 1) systemic arthritis, 2)polyarthritis RF positive, 3) polyarthritis RF negative, 4)oligoarthritis, 5) extended oligoarthritis, 6) enthesitis relatedarthritis, 7) psoriatic arthritis, and 8) others.

Furthermore, the present inventors have proposed a method of classifyingchronic arthritides diseases of childhood into:

1) primary chronic arthritides of childhood

(1) SPRASH syndrome (SPRASH: spiking fever, pericarditis, rash,arthritis, splenomegaly, hepatomegaly)

Starts with relaxation heat and efflorescence, and serositis andhepatomegaly are observed with concomitant onset of simultaneous ordelayed arthritis, but at times arthritis may not be observed.

(2) idiopathic chronic arthritides of childhood

No underlying diseases are present, and arthritis is the key pathology.

-   -   a) rheumatoid factor (RF)-positive type    -   b) anti-nuclear antibody (ANA)-positive type    -   c) RF/ANA-negative type

2) secondary chronic arthritides of childhood

Genetic or nongenetic original diseases are accompanied by arthritis(Shunpei Yokota, “Advances in recent therapeutic methods for chronicarthritides diseases of childhood”, Rheumatism, 39: 860 (1999)).

It has been reported that various cytokines are involved in chronicarthritides diseases of childhood. In particular, it is thought thatimbalance in inflammatory cytokines IL-1, IL-6, IL-12 and TNF-α, andanti-inflammatory cytokines IL-1ra (IL-1 receptor antagonist), IL-10,IL-13, sTNFR (soluble TNF receptor) is associated with the disease.

For the treatment of chronic arthritides diseases of childhood,nonsteroidal anti-inflammatory drugs, corticosteroids, antirheumaticdrugs (gold compounds etc.), immunosuppressants, methotrexate (MTX etc.)have been used. However, as the therapeutic effects differ with thepatients, the development of more effective therapeutic regimens isbeing awaited.

Still's disease, first described by the British pediatrician Dr. Stillin 1897, was reported to have a clinical picture clearly different fromthat of rheumatoid arthritis in adults and is a disease seen in childrento adults (especially in adolescence and the main symptoms includefever, erythema, arthritis, serositis and the like. Among them,adult-onset type is designated as adult onset Still's disease. InStill's disease, rheumatoid factor is usually negative.

In children, Still's disease is another name of the systemic type ofjuvenile rheumatoid arthritis (juvenile rheumatoid arthritis (JRA), JCA(juvenile chronic arthritis), juvenile idiopathic arthritis (JIA)) whichis a chronic arthritis developing in children at less than 16 years old.For the causes of Still's disease, environmental factors such as avirus, host factors such as HLA, and immunological abnormalities havebeen reported, but the etiology is still obscure.

Still's disease in adults and that in children are considered to bealmost the same disease, though there are minor differences in clinicalfeature in addition to the age when the disease develops. Still'sdisease in children refers to JRA of the systemic type as describedabove. However, JRA and rheumatoid arthritis (RA) in adults areclinically different in many ways and are dealt with as differentdiseases, and therefore Still's disease in adults is often dealt with asan independent disease entity among the rheumatic diseases.

As diagnostic criteria for Still's disease in adults, there have beenknown those by Yamaguchi (Journal of Rheumatology 19(3): 424-30, 1992),Reginato (Seminars in Arthritis & Rheumatism 17(1): 39-57, 1987), Cush(Rheumatology Grand Rounds, University of Pittsburgh Medical Center;Jan. 30, 1984), Goldman (Southern Medical Journal 73: 555-563, 1980) andthe like.

On the relationship between Still's disease and cytokines, associationwith cytokines such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, TNF-α,and IFN-γ has been reported, and among them, inflammatory cytokines suchas IL-1, IL-6, TNF-α, and IFN-γ have been implicated in the pathology ofStill's disease.

With respect to IL-6, de Benedetti et al. reported that serum levels ofIL-6 are elevated in Still's disease in children (Arthritis Rheum. 34:1158, 1991), and that a large amount of IL-6/soluble IL-6 receptor(sIL-6R) complex is present in the serum of patients with Still'sdisease in children and a correlation can be seen between this complexlevel and CRP values (J. Clin. Invest. 93: 2114, 1994). Furthermore,Rooney et al. have reported that plasma levels of IL-6 and TNF-α areelevated in patients with Still's disease in children (Br. J. Rheumatol.34: 454, 1995).

As a method of treating Still's disease, nonsteroidal anti-inflammatorydrugs, corticosteroids, antirheumatic drugs (gold compounds etc.),immunosuppressants, gamma globulin formulations, methotrexate (MTX etc.)have been used. However, as the therapeutic effects differ with thepatients, the development of more effective therapeutic regimens isbeing sought after.

DISCLOSURE OF THE INVENTION

Thus, the present invention provides a novel therapeutic agent forchronic arthritides diseases of childhood-related diseases, said agentbeing of a type different from the conventional therapeutic agents forchronic arthritides diseases of childhood-related diseases. Inaccordance with the present invention, chronic arthritides diseases ofchildhood-related diseases included chronic arthritides diseases ofchildhood and Still's disease.

After intensive and extensive study to solve the above problems, thepresent inventors have found that an interleukin-6 (IL-6) antagonist hasan effect of treating chronic arthritides diseases of childhood-relateddiseases, and have completed the present invention.

Thus, the present invention provides a therapeutic agent for chronicarthritides diseases of childhood-related diseases comprising aninterleukin-6 (IL-6) antagonist as an active ingredient.

More specifically, the present invention provides a therapeutic agentfor chronic arthritides diseases of childhood comprising aninterleukin-6 (IL-6) antagonist as an active ingredient.

The present invention also provides a therapeutic agent for Still'sdisease comprising an interleukin-6 (IL-6) antagonist as an activeingredient.

BEST MODE FOR CARRYING OUT THE INVENTION

The above IL-6 antagonist is preferably an antibody against IL-6receptor, and preferably a monoclonal antibody against human IL-6receptor or a monoclonal antibody against mouse IL-6 receptor. As theabove monoclonal antibody against human IL-6 receptor, there can beillustrated PM-1 antibody, and as the above monoclonal antibody againstmouse IL-6 receptor, there can be illustrated MR16-1 antibody.

The above antibody is preferably a chimeric antibody, a humanizedantibody or a human antibody and, for example, is a humanized PM-1antibody.

Chronic arthritides diseases of childhood which are the subject oftreatment with a therapeutic agent of the present invention include alldiseases in the above ARA, EULAR, and ILAR classifications, and theclassification by the present inventors. With the advance in theserological diagnostic methods and the advance in therapeutic methods,the disease type classification of chronic arthritides diseases ofchildhood is now undergoing a review on a global scale and it can besaid to be in a state of uncertainty. Preferred treatment subjects, forthe therapeutic agent of the present invention, are: in the ARAclassification, systemic onset, polyarticular, and pauciarticular; inthe EULAR classification, systemic onset, polyarticular, andoligoarticular; in the ILAR classification, systemic onset,polyarticular (RF positive), polyarticular (RF negative),oligoarthritis, and extended oligoarthritis; and, in the classificationby the present inventors, primary chronic arthritides of childhood(SPRASH syndrome, idiopathic chronic arthritides of childhood (a.rheumatoid factor (RF)-positive type, b. anti-nuclear antibody(ANA)-positive type, c. RF/ANA-negative type)), and as most preferredsubjects of treatment are: in the ARA classification, systemic onset andpolyarticular; in the EULAR classification, systemic onset andpolyarticular; in the ILAR classification, systemic onset, polyarticular(RF positive), polyarticular (RF negative), and extended oligoarthritis;and, in the classification by the present inventors, primary chronicarthritides of childhood (SPRASH syndrome, idiopathic chronicarthritides of childhood (a. rheumatoid factor (RF)-positive type, b.anti-nuclear antibody (ANA)-positive type)). More preferred subjects oftreatment are: in the ARA classification, systemic onset andpolyarticular; in the EULAR classification, systemic onset andpolyarticular; in the ILAR classification, systemic onset, polyarticular(RF positive), and extended oligoarthritis; and, in the classificationby the present inventors, primary chronic arthritides of childhood(SPRASH syndrome, idiopathic chronic arthritides of childhood (a.rheumatoid factor (RF)-positive type)).

IL-6 antagonists for use in the present invention may be of any origin,any type, and any form, as long as they exhibit therapeutic effects onchronic arthritides diseases of childhood-related diseases.

IL-6 antagonists are substances that block signal transduction by IL-6and inhibit the biological activity of IL-6. IL-6 antagonists aresubstances that preferably have an inhibitory action on the binding toany of IL-6, IL-6 receptor or gp130. As IL-6 antagonists, there can bementioned, for example, anti-IL-6 antibody, anti-IL-6 receptor antibody,anti-gp130 antibody, reshaped IL-6, soluble reshaped IL-6 receptor, orpartial peptides of IL-6 or IL-6 receptor, as well as low molecularweight substances that exhibit activities similar to them.

Anti-IL-6 antibodies for use in the present invention can be obtained aspolyclonal or monoclonal antibodies using a known method. As theanti-IL-6 antibodies for use in the present invention, monoclonalantibodies of, in particular, mammalian origin are preferred. Monoclonalantibodies of a mammalian origin include those produced by a hybridomaand those produced by a host which has been transformed by geneengineering technology with an expression vector containing the antibodygene. These antibodies, via binding to IL-6, block the binding of IL-6to IL-6 receptor, and thereby block the propagation of biologicalactivity of IL-6 into the cell.

Examples of such antibodies include MH166 antibody (Matsuda, et al.,Eur. J. Immunology (1988) 18, 951-956), or SK2 antibody (Sato, et al.,The 21st General Meeting of the Japanese Society for Immunology,Gakujutu Kiroku (1991) 21, 166) etc.

A hybridoma that produces anti-IL-6 antibody can be basicallyconstructed using a known procedure as described bellow. Thus, IL-6 isused as a sensitizing antigen, which is immunized in the conventionalmethod of immunization, and the immune cells thus obtained are fusedwith known parent cells in a conventional cell fusion process, followedby a conventional screening method to screen monoclonalantibody-producing cells.

Specifically, anti-IL-6 antibodies may be obtained in the followingmanner. For example, human IL-6 to be used as the sensitizing antigenfor obtaining antibody can be obtained using the IL-6 gene/amino acidsequence disclosed in Eur. J. Biochem. (1987) 168, 543-550; J. Immunol.(1988) 140, 1534-1541, or Agr. Biol. Chem. (1990) 54, 2685-2688.

After the gene sequence of IL-6 was inserted into a known expressionvector to transform a suitable host cell, the IL-6 protein of interestmay be purified from the host cell or a culture supernatant thereof by aknown method, and the purified IL-6 protein may be used as thesensitizing antigen. Alternatively, a fusion protein of the IL-6 proteinand another protein may be used as the sensitizing antigen.

Anti-IL-6 receptor antibodies for use in the present invention can beobtained as polyclonal or monoclonal antibodies using a known method. Asthe anti-IL-6 receptor antibodies for use in the present invention,monoclonal antibodies of, in particular, a mammalian origin arepreferred. Monoclonal antibodies of a mammalian origin include thoseproduced by a hybridoma and those produced by a host which has beentransformed by gene engineering technology with an expression vectorcontaining the antibody gene. These antibodies, via binding to IL-6,block the binding of IL-6 to IL-6 receptor, and thereby block thepropagation of biological activity of IL-6 into the cell.

Examples of such antibodies include MR16-1 antibody (Tamura, T. et al.,Proc. Natl. Acad. Sci. USA (1993) 90, 11924-11928), PM-1 antibody(Hirata, Y. et al., J. Immunology (1989) 143, 2900-2906), AUK12-20antibody, AUK64-7 antibody or AUK146-15 antibody (International PatentApplication WO 92-19759), and the like. Among them, PM-1 antibody ismost preferred.

Incidentally, the hybridoma cell line which produces PM-1 antibody hasbeen internationally deposited under the provisions of the BudapestTreaty as PM-1 on Jul. 12, 1988 with the International Patent OrganismDepository of the National Institute of Industrial Science andTechnology (Central 6, 1-1-1 Higashi, Tsukuba City, Ibaraki Pref.,305-5466 Japan) as FERM BP-2998. Also, the hybridoma cell line whichproduces MR16-1 antibody has been internationally deposited under theprovisions of the Budapest Treaty as Rat-mouse hybridoma MR16-1 on Mar.13, 1997 with the International Patent Organism Depository of theNational Institute of Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba City, Ibaraki Pref., 305-5466 Japan) as FERMBP-5875.

A hybridoma that produces anti-IL-6 receptor monoclonal antibody can,basically, be constructed using a known procedure as described bellow.Thus, IL-6 receptor is used as a sensitizing antigen, which is immunizedin the conventional method of immunization, and the immune cells thusobtained are fused with known parent cells in a conventional cell fusionprocess, followed by a conventional screening method to screenmonoclonal antibody-producing cells.

Specifically, anti-IL-6 receptor antibodies may be obtained in thefollowing manner. For example, human IL-6 receptor used as thesensitizing antigen for obtaining antibody can be obtained using theIL-6 receptor gene/amino acid sequence disclosed in European PatentApplication No. EP 325474, and mouse IL-6 receptor can be obtained usingthe IL-6 receptor gene/amino acid sequence disclosed in JapaneseUnexamined Patent Publication (Kokai) No. 3-155795.

There are two types of IL-6 receptor: IL-6 receptor expressed on thecell membrane, and IL-6 receptor detached from the cell membrane(Soluble IL-6 Receptor; Yasukawa et al., J. Biochem. (1990) 108,673-676). Soluble IL-6 receptor antibody is composed of thesubstantially extracellular region of IL-6 receptor bound to the cellmembrane, and is different from the membrane-bound IL-6 receptor in thatthe former lacks the transmembrane region or both of the transmembraneregion and the intracellular region. IL-6 receptor protein may be anyIL-6 receptor, as long as it can be used as a sensitizing antigen forpreparing anti-IL-6 receptor antibody for use in the present invention.

After a gene encoding IL-6 receptor has been inserted into a knownexpression vector system to transform an appropriate host cell, thedesired IL-6 receptor protein may be purified from the host cell or aculture supernatant thereof using a known method, and the IL-6 receptorprotein thus purified may be used as the sensitizing antigen.Alternatively, cells that express IL-6 receptor protein or a fusionprotein of IL-6 receptor protein and another protein may be used as thesensitizing antigen.

Escherichia coli (E. coli) containing a plasmid pIBIBSF2R that comprisescDNA encoding human IL-6 receptor has been internationally depositedunder the provisions of the Budapest Treaty as HB101-pIBIBSF2R on Jan.9, 1989 with the International Patent Organism Depository of theNational Institute of Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba City, Ibaraki Pref., 305-5466 Japan) as FERMBP-2232.

Anti-gp130 antibodies for use in the present invention can be obtainedas polyclonal or monoclonal antibodies using a known method. As theanti-gp130 antibodies for use in the present invention, monoclonalantibodies of, in particular, mammalian origin are preferred. Monoclonalantibodies of a mammalian origin include those produced by a hybridomaand those produced by a host which has been transformed by geneengineering technology with an expression vector containing the antibodygene. These antibodies, via binding to gp130, block the binding of gp130to the IL-6/IL-6 receptor complex, and thereby block the propagation ofbiological activity of IL-6 into the cell.

Examples of such antibodies include AM64 antibody (Japanese UnexaminedPatent Publication (Kokai) No. 3-219894), 4B11 antibody and 2H4 antibody(U.S. Pat. No. 5,571,513), B-S12 antibody and B-P8 antibody (JapaneseUnexamined Patent Publication (Kokai) No. 8-291199) etc.

A hybridoma that produces anti-gp130 antibody can be basicallyconstructed using a known procedure as described below. Thus, gp130 isused as a sensitizing antigen, which is immunized in the conventionalmethod of immunization, and the immune cells thus obtained are fusedwith known parent cells in a conventional cell fusion process, followedby a conventional screening method to screen monoclonalantibody-producing cells.

Specifically, monoclonal antibodies may be obtained in the followingmanner. For example, gp130 used as the sensitizing antigen for obtainingantibody can be obtained using the gp130 gene/amino acid sequencedisclosed in European Patent Application No. EP 411946.

The gene sequence of gp130 may be inserted into a known expressionvector, and said vector is used to transform a suitable host cell. Fromthe host cell or a culture supernatant therefrom, the gp130 protein ofinterest may be purified by a known method, and the purified IL-6protein may be used as the sensitizing antigen. Alternatively, cellsexpressing gp130, or a fusion protein of the gp130 protein and anotherprotein may be used as the sensitizing antigen.

Preferably, mammals to be immunized with the sensitizing antigen areselected in consideration of their compatibility with the parent cellsfor use in cell fusion and they generally include, but are not limitedto, rodents such as mice, rats and hamsters.

Immunization of animals with a sensitizing antigen is carried out usinga known method. A general method, for example, involves intraperitonealor subcutaneous administration of a sensitizing antigen to the mammal.Specifically, a sensitizing antigen, which was diluted and suspended inan appropriate amount of phosphate buffered saline (PBS) orphysiological saline etc., is mixed with an appropriate amount of acommon adjuvant such as Freund's complete adjuvant. After beingemulsified, it is preferably administered to a mammal several timesevery 4 to 21 days. Additionally, a suitable carrier may be used at thetime of immunization of the sensitizing antigen.

After the immunization and confirmation of an increase in the desiredantibody levels in the serum by a conventional method, immune cells aretaken out from the mammal and are subjected to cell fusion. As preferredimmune cells that are subjected to cell fusion, there can bespecifically mentioned spleen cells.

Mammalian myeloma cells as the other parent cells which are subjected tocell fusion with the above-mentioned immune cells preferably includevarious known cell lines such as P3x63Ag8.653 (Kearney, J. F. et al., J.Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics inMicrobiology and Immunology (1978) 81, 1-7), NS-1 (Kohler, G. andMilstein, C., Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies, D.H. et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M. et al., Nature(1978) 276, 269-270), FO (de St. Groth, S. F. et al., J. Immunol.Methods (1980) 35, 1-21), S194 (Trowbridge, I. S., J. Exp. Med. (1978)148, 313-323), R210 (Galfre, G. et al., Nature (1979) 217, 131-133) andthe like, which may be used as appropriate.

Cell fusion between the above immune cells and myeloma cells may beessentially conducted in accordance with a known method such as isdescribed in Milstein et al. (Kohler, G. and Milstein, C., MethodsEnzymol. (1981) 73, 3-46) and the like.

More specifically, the above cell fusion is carried out in theconventional nutrient broth in the presence of, for example, a cellfusion accelerator. As the cell fusion accelerator, for example,polyethylene glycol (PEG), Sendai virus (HVJ) and the like may be used,and an adjuvant such as dimethyl sulfoxide may be added as desired toenhance the efficiency of fusion.

The preferred ratio of the immune cells and the myeloma cells for useis, for example, 1 to 10 times more immune cells than the myeloma cells.Examples of culture media to be used for the above cell fusion include,for example, RPMI 1640 medium and MEM culture medium suitable for thegrowth of the above myeloma cell lines, and the conventional culturemedium used for this type of cell culture and, besides, a serumsupplement such as fetal calf serum (FCS) may be added.

In cell fusion, predetermined amounts of the above immune cells and themyeloma cells are mixed well in the above culture liquid, to which a PEGsolution previously heated to about 37° C., for example a PEG solutionwith a mean molecular weight of 1000 to 6000, is added at aconcentration of 30 to 60% (w/v) and mixed to obtain the desired fusioncells (hybridomas). Then, by repeating a sequential addition of asuitable culture liquid and centrifugation to remove the supernatant,cell fusion agents etc., that are undesirable for the growth of thehybridoma, can be removed.

Said hybridoma is selected by culturing in the conventional selectionmedium, for example, HAT culture medium (a culture liquid containinghypoxanthine, aminopterin, and thymidine). Culturing in said HAT culturemedium is continued generally for the period of time sufficient toeffect killing of the cells other than the desired hybridoma (non-fusioncells), generally several days to several weeks. The conventionallimiting dilution method is conducted in which the hybridomas producingthe desired antibody are screened and cloned.

In addition to obtaining the above hybridoma by immunizing an animalother than the human with an antigen, it is also possible to sensitizehuman lymphocytes in vitro with the desired antigen protein orantigen-expressing cells, and the resulting sensitized B-lymphocytes arefused with a myeloma cell, for example U266, having the ability ofdividing permanently to obtain a hybridoma that produces the desiredhuman antibody having the activity of binding to the desired antigen orantigen-expressing cells (Japanese Post-examined Patent Publication(Kokoku) 1-59878). Furthermore, a transgenic animal having a repertoireof human antibody genes is immunized with the antigen orantigen-expressing cells to obtain the desired human antibody accordingto the above-mentioned method (see International Patent Application WO93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO96/33735).

The monoclonal antibody-producing hybridomas thus constructed can besubcultured in the conventional culture liquid, or can be stored for aprolonged period of time in liquid nitrogen.

In order to obtain monoclonal antibodies from said hybridoma, there canbe used a method in which said hybridoma is cultured in the conventionalmethod and the antibodies are obtained as the supernatant, or a methodin which the hybridoma is implanted into and grown in a mammalcompatible with said hybridoma and the antibodies are obtained as theascites. The former method is suitable for obtaining high-purityantibodies, whereas the latter is suitable for a large scale productionof antibodies.

For example, an anti-IL-6 receptor antibody-producing hybridoma can bepolypeptide by a method disclosed in Japanese Unexamined PatentPublication (Kokai) No. 3-139293. There may be used a method in whichThe PM-1 antibody-producing hybridoma that has been internationallydeposited under the provisions of the Budapest Treaty on Jul. 12, 1988with the International Patent Organism Depository of the NationalInstitute of Industrial Science and Technology (Central 6, 1-1-1Higashi, Tsukuba City, Ibaraki Pref., 305-5466 Japan) as FERM BP-2998 isintraperitoneally injected to BALB/c mice to obtain ascites, from whichascites PM-1 antibody may be purified, or a method in which thehybridoma is cultured in a RPMI 1640 medium containing 10% bovine fetalserum, 5% BM-Codimed H1 (manufactured by Boehringer Mannheim), thehybridoma SFM medium (manufactured by GIBCO BRL), the PFHM-II medium(manufactured by GIBCO BRL) or the like, from the culture supernatant ofwhich PM-1 antibody may be purified.

In accordance with the present invention, as monoclonal antibody, therecan be used a recombinant antibody that was produced by cloning anantibody gene from a hybridoma and the gene is then integrated into anappropriate vector, which is introduced into a host to produce therecombinant antibody using gene recombinant technology (see, forexample, Borrebaeck, C. A. K. and Larrick, J. W., THERAPEUTIC MONOCLONALANTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD.1990).

Specifically, mRNA encoding the variable region (V region) of theantibody is isolated from the cell that produces the antibody ofinterest, for example a hybridoma. The isolation of mRNA is conducted bypreparing total RNA by a known method such as the guanidineultracentrifuge method (Chirgwin, J. M. et al., Biochemistry (1979) 18,5294-5299), the AGPC method (Chomczynski, P. et al., Anal. Biochem.(1987) 162, 156-159), and then mRNA is purified from the total RNA usingthe mRNA Purification kit (manufactured by Pharmacia) and the like.Alternatively, mRNA can be directly prepared using the Quick Prep mRNAPurification Kit (manufactured by Pharmacia).

cDNA of the V region of antibody may be synthesized from the mRNA thusobtained using a reverse transcriptase. cDNA may be synthesized usingthe AMV Reverse Transcriptase First-strand cDNA Synthesis Kit and thelike. Alternatively, for the synthesis and amplification of cDNA, the5′-Ampli FINDER RACE Kit (manufactured by Clontech) and the 5′-RACEmethod (Frohman, M. A. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17,2919-2932) which employs PCR may be used. The desired DNA fragment ispurified from the PCR product obtained and may be ligated to vector DNA.Moreover, a recombinant vector is constructed therefrom and then isintroduced into E. coli etc., from which colonies are selected toprepare the desired recombinant vector. The base sequence of the desiredDNA may be confirmed by a known method such as the dideoxy method.

Once DNA encoding the V region of the desired antibody has beenobtained, it may be ligated to DNA encoding the constant region (Cregion) of the desired antibody, which is then integrated into anexpression vector. Alternatively, DNA encoding the V region of theantibody may be integrated into an expression vector which alreadycontains DNA encoding the C region of the antibody.

In order to produce antibody for use in the present invention, theantibody gene is integrated into an expression vector so as to beexpressed under the control of the expression regulatory region, forexample an enhancer and/or a promoter. Subsequently, the expressionvector is transformed into a host cell and the antibody can then beexpressed therein.

In accordance with the present invention, artificially alteredrecombinant antibodies such as chimeric antibody, humanized antibody andhuman antibody can be used for the purpose of lowering heterologousantigenicity against humans. These altered antibody can be producedusing known methods.

Chimeric antibody can be obtained by ligating the thus obtained DNAencoding the V region of antibody to DNA encoding the C region of humanantibody, which is then integrated into an expression vector andintroduced into a host for production of the antibody therein (seeEuropean Patent Application EP 125023, and International PatentApplication WO 92-19759). Using this known method, chimeric antibodyuseful for the present invention can be obtained.

Plasmids containing the L chain V region or the H chain V region ofchimeric PM-1 antibody have each been designated as pPM-k3 and pPM-h1,respectively, and E. coli having a respective plasmid has beeninternationally deposited under the provisions of the Budapest Treaty asNCIMB40366 and NCIMB40362 on Feb. 11, 1991 with the National Collectionsof Industrial and Marine Bacteria Limited.

Humanized antibody which is also called reshaped human antibody has beenmade by implanting the complementarity determining region (CDR) ofantibody of a mammal other than the human, for example mouse antibody,into the CDR of human antibody. The general recombinant DNA technologyfor preparation of such antibodies is also known (see European PatentApplication EP 125023 and International Patent Application WO 92-19759).

Specifically, a DNA sequence which was designed to ligate the CDR ofmouse antibody with the framework region (FR) of human antibody issynthesized from several divided oligonucleotides having sectionsoverlapping with one another at the ends thereof. The DNA thus obtainedis ligated to DNA encoding the C region of human antibody and then isincorporated into an expression vector, which is introduced into a hostfor antibody production (see European Patent Application EP 239400 andInternational Patent Application WO 92-19759).

For the FR of human antibody ligated through CDR, the CDR that has afavorable antigen-binding site is selected. When desired, amino acids inthe FR of antibody V region may be substituted so that the CDR ofhumanized antibody may form an appropriate antigen biding site (Sato, K.et al., Cancer Res. (1993) 53, 851-856).

As the C region of human antibody, there can be used, for example, Cγ1,Cγ2, Cγ3, or Cγ4. The C region of human antibody may also be modified inorder to improve the stability of antibody and of the productionthereof.

Chimeric antibody consists of the V region of antibody of a human originother than humans and the C region of human antibody, and humanizedantibody consists of the complementarity determining region of antibodyof a human origin other than humans and the framework region and the Cregion of human antibody, with their antigenicity in the human bodybeing decreased, and thus are useful as antibody for use in the presentinvention.

As a preferred embodiment of humanized antibody for use in the presentinvention, there can be mentioned humanized PM-1 antibody (seeInternational Patent Application WO 92-19759).

As a method of obtaining human antibody, in addition to those describedabove, there is known a method of obtaining human antibody by means ofpanning. For example, the variable region of human antibody is expressedon the surface of a phage by the phage display method as a single chainantibody (scFv) to select a phage that binds to the antigen. Byanalyzing the gene of the phage selected, the DNA sequence encoding thevariable region of the human antibody that binds to the antigen can beidentified. Once the DNA sequence of scFv that binds to the antigen hasbeen clarified, said sequence can be used to prepare a suitableexpression vector and human antibody can be obtained. These methods arealready known and can be found in WO 92/01047, WO 92/20791, WO 93/06213,WO 93/11236, WO 93/19172, WO 95/01438, and WO 95/15388.

Antibody genes constructed as mentioned above may be expressed andobtained in a known manner. In the case of mammalian cells, expressionmay be accomplished using a DNA in which a commonly used usefulpromoter, an antibody gene to be expressed, and the poly A signal havebeen operably linked at 3′ downstream thereof, or a vector containingit. As the promoter/enhancer, for example, there can be mentioned humancytomegalovirus immediate early promoter/enhancer.

Additionally, as the promoter/enhancer which can be used for expressionof antibody for use in the present invention, there can be used viralpromoters/enhancers such as retrovirus, polyoma virus, adenovirus, andsimian virus 40 (SV40), and promoters/enhancers derived from mammaliancells such as human elongation factor 1α (HEF1α).

For example, expression may be readily accomplished by the method ofMulligan et al. (Mulligan, R. C. et al., Nature (1979) 277, 108-114)when SV40 promoter/enhancer is used, and by the method of Mizushima, S.et al. (Mizushima, S. and Nagata, S., Nucleic Acids Res. (1990) 18,5322) when HEF1α promoter/enhancer is used.

In the case of E. coli, expression may be conducted by operably linkinga commonly used promoter, a signal sequence for antibody secretion, andan antibody gene to be expressed, followed by expression thereof. As thepromoter, for example, there can be mentioned lacz promoter and araBpromoter. The method of Ward et al. (Ward, E. S. et al., Nature (1989)341, 544-546; Ward, E. S. et al., FASEB J. (1992) 6, 2422-2427) may beused when lacz promoter is used, and the method of Better et al.(Better, M. et al., Science (1988) 240, 1041-1043) may be used when araBpromoter is used.

As a signal sequence for antibody secretion, when produced in theperiplasm of E. coli, the pelB signal sequence (Lei, S. P. et al., J.Bacteriol. (1987) 169, 4379-4383) can be used. After separating theantibody produced in the periplasm, the structure of the antibody isappropriately refolded before use (see, for example, WO 96-30394).

As the origin of replication, there can be used those derived from SV40,polyoma virus, adenovirus, bovine papilloma virus (BPV), and the like.Furthermore, for amplification of the gene copy number in the host cellsystem, expression vectors can include, as selectable markers theaminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene,E. coli xanthine guaninephosphoribosyl transferase (Ecogpt) gene, thedihydrofolate reductase (dhfr) gene, and the like.

For the production of antibody for use in the present invention, anyproduction system can be used, and the production systems of antibodypreparation comprise the in vitro or the in vivo production system. Asthe in vitro production systems, there can be mentioned a productionsystem which employs eukaryotic cells and the production system whichemploys prokaryotic cells.

When eukaryotic cells are used, there are the production systems whichemploy animal cells, plant cells, and fungal cells. Known animal cellsinclude (1) mammalian cells such as CHO cells, COS cells, myeloma cells,baby hamster kidney (BHK) cells, HeLa cells, and Vero cells, (2)amphibian cells such as Xenopus oocytes, or (3) insect cells such assf9, sf21, and Tn5. Known plant cells include, for example, thosederived from the Nicotiana tabacum which is subjected to callus culture.Known fungal cells include yeasts such as genus Saccharomyces, morespecifically Saccharomyces cereviceae, or filamentous fungi such as theAspergillus family, more specifically Aspergillus niger.

When prokaryotic cells are used, there are the production systems whichemploy bacterial cells. Known bacterial cells include Escherichia coli,and Bacillus subtilis.

By introducing, via transformation, the gene of the desired antibodyinto these cells and culturing the transformed cells in vitro, theantibody can be obtained. Culturing is conducted in the known methods.For example, as the culture liquid for mammalian cells, DMEM, MEM,RPMI1640, IMDM and the like can be used, and serum supplements such asfetal calf serum (FCS) may be used in combination. In addition,antibodies may be produced in vivo by implanting cells into which theantibody gene has been introduced into the abdominal cavity of ananimal, and the like.

As in vivo production systems, there can be mentioned those which employanimals and those which employ plants. When animals are used, there arethe production systems which employ mammals and insects.

As mammals, goats, pigs, sheep, mice, and cattle can be used (VickiGlaser, SPECTRUM Biotechnology Applications, 1993). Also, as insects,silkworms can be used and, in the case of plants, tobacco, for example,can be used.

Antibody genes are introduced into these animals and plants, in whichthe genes are produced and then collected. For example, antibody genesare inserted into the middle of the gene encoding protein which isinherently produced in the milk such as goat β casein to prepare fusiongenes. DNA fragments containing the fusion gene into which the antibodygene has been inserted are injected to a goat embryo, and the embryo isintroduced into a female goat. The desired antibody is obtained from themilk produced by a transgenic goat produced by the goat that receivedthe embryo or the offspring thereof. In order to increase the amount ofmilk containing the desired antibody produced by the transgenic goat,hormones may be given to the transgenic goat as appropriate (Ebert, K.M. et al., Bio/Technology (1994) 12, 699-702).

When silkworms are used, the silkworm is infected with a baculovirusinto which desired antibody gene has been inserted, and the desiredantibody can be obtained from the body fluid of the silkworm (Maeda, S.et al., Nature (1985) 315, 592-594). Moreover, when tobacco is used, thedesired antibody gene is inserted into an expression vector for plants,for example pMON 530, and then the vector is introduced into a bacteriumsuch as Agrobacterium tumefaciens. The bacterium is then used to infecttobacco such as Nicotiana tabacum to obtain the desired antibody fromthe leaves of the tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol.(1994) 24, 131-138).

When antibody is produced in an in vitro or in vivo production systems,as mentioned above, DNA encoding the heavy chain (H chain) or lightchain (L chain) of antibody is separately incorporated into anexpression vector and the hosts are transformed simultaneously, or DNAencoding the H chain and the L chain of antibody is integrated into asingle expression vector and the host is transformed therewith (seeInternational Patent Application WO 94-11523).

Antibodies for use in the present invention may be fragments of antibodyor modified versions thereof as long as they are preferably used in thepresent invention. For example, as fragments of antibody, there may bementioned Fab, F(ab′)2, Fv or single-chain Fv (scFv) in which Fv's of Hchain and L chain were ligated via a suitable linker.

Specifically antibodies are treated with an enzyme, for example, papainor pepsin, to produce antibody fragments, or genes encoding theseantibody fragments are constructed, and then introduced into anexpression vector, which is expressed in a suitable host cell (see, forexample, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M.and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Plucktrun, A.and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E.,Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., MethodsEnzymol. (1986) 121, 663-669; Bird, R. E. et al., TI BTECH (1991) 9,132-137).

scFv can be obtained by ligating the V region of H chain and the Vregion of L chain of antibody. In the scFv, the V region of H chain andthe V region of L chain are preferably ligated via a linker, preferablya peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A.(1988) 85, 5879-5883). The V region of H chain and the V region of Lchain in the scFv may be derived from any of the above-mentionedantibodies. As the peptide linker for ligating the V regions, anysingle-chain peptide comprising, for example, 12-19 amino acid residuesmay be used.

DNA encoding scFv can be obtained using DNA encoding the H chain or theH chain V region of the above antibody and DNA encoding the L chain orthe L chain V region of the above antibody as the template by amplifyingthe portion of the DNA encoding the desired amino acid sequence amongthe above sequences by the PCR technique with the primer pair specifyingthe both ends thereof, and by further amplifying the combination of DNAencoding the peptide linker portion and the primer pair which definesthat both ends of said DNA be ligated to the H chain and the L chain,respectively.

Once DNAs encoding scFv are constructed, an expression vector containingthem and a host transformed with said expression vector can be obtainedby a conventional method, and scFv can be obtained using the resultanthost by a conventional method.

These antibody fragments can be produced by obtaining the gene thereofin a similar manner to that mentioned above, and by allowing it to beexpressed in a host. “Antibody” as used in the present inventionencompasses these antibody fragments.

As modified antibodies, antibodies associated with various moleculessuch as polyethylene glycol (PEG) can be used. “Antibody” as used in thepresent invention encompasses these modified antibodies. These modifiedantibodies can be obtained by chemically modifying the antibodies thusobtained. These methods have already been established in the art.

Antibodies expressed and produced as described above can be separatedfrom inside or outside of the cell or from the host and then may bepurified to homogeneity. Separation and purification of antibody for usein the present invention may be accomplished by affinity chromatography.As the column used for affinity chromatography, there can be mentionedProtein A column and Protein G column. Examples of carriers for use inProtein A column include, for example, Hyper D, POROS, Sepharose F.F.(Pharmacia) and the like. In addition, commonly used methods ofseparation and purification for proteins can be used, without anylimitation.

Chromatography other than the above affinity chromatography, filters,gel filtration, salting out, dialysis and the like may be selected andcombined as appropriate, in order to separate and purify the antibodiesfor use in the present invention. Chromatography includes, for example,ion exchange chromatography, hydrophobic chromatography, gel-filtrationand the like. These chromatographies can be applied to high performanceliquid chromatography (HPLC). Also, reverse phase HPLC (rpHPLC) may beused.

The concentration of antibody obtained as above can be determined bymeasurement of absorbance or by ELISA and the like. Thus, whenabsorbance measurement is employed, the antibody obtained isappropriately diluted with PBS(-) and then the absorbance is measured at280 nm, followed by calculation using the absorption coefficient of 1.35OD at 1 mg/ml. When ELISA is used, measurement is conducted as follows.Thus, 100 μl of goat anti-human IgG antibody (manufactured by TAGO)diluted to 1 μg/ml in 0.1 M bicarbonate buffer, pH 9.6, is added to a96-well plate (manufactured by Nunc), and is incubated overnight at 4°C. to immobilize the antibody. After blocking, 100 μl each ofappropriately diluted antibody for use in the present invention orsamples containing the antibody, or human IgG (manufactured by CAPPEL)as the standard is added, and incubated at room temperature for 1 hour.

After washing, 100 μl of 5000-fold diluted alkaline phosphatase-labeledanti-human IgG antibody (manufactured by BIO SOURCE) is added, andincubated at room temperature for 1 hour. After washing, the substratesolution is added and incubated, followed by measurement of absorbanceat 405 nm using the MICROPLATE READER Model 3550 (manufactured byBio-Rad) to calculate the concentration of the desired antibody.

Reshaped IL-6 for use in the present invention is a substance that hasan activity of binding with IL-6 receptor and that does not propagatethe biological activity of IL-6. Thus, though reshaped IL-6 competeswith IL-6 for binding to IL-6 receptor, it does not propagate thebiological activity of IL-6, and therefore reshaped IL-6 blocks signaltransduction by IL-6.

Reshaped IL-6 may be prepared by introducing mutations by replacingamino acid residues of the amino acid sequence of IL-6. IL-6 from whichreshaped IL-6 is derived may be of any origin, but it is preferablyhuman IL-6 considering antigenicity etc.

Specifically, the secondary structure of the amino acid sequence of IL-6may be estimated using a known molecular modeling program such as WHATIF(Vriend et al., J. Mol. Graphics (11990) 8, 52-56), and its effect onthe overall amino acid residues to be replaced is evaluated. Afterdetermining suitable amino acid residues, mutation may be introducedusing a vector containing a base sequence encoding human IL-6 gene as atemplate in a commonly used PCR method so as to replace amino acids, andthereby to obtain a gene encoding reshaped IL-6. This may be integrated,as appropriate, into a suitable expression vector to obtain reshapedIL-6 according to the above-mentioned methods for expression,production, and purification of recombinant antibody.

Specific examples of reshaped IL-6 has been disclosed in Brakenhoff etal., J. Biol. Chem. (1994) 269, 86-93, Saviono et al., EMBO J. (1994)13, 1357-1367, WO 96-18648 and WO 96-17869.

Partial peptides of IL-6 or partial peptides of IL-6 receptor for use inthe present invention are substances that have an activity of binding toIL-6 receptor or IL-6, respectively, and that do not propagate thebiological activity of IL-6. Thus, partial peptides of IL-6 or partialpeptides of IL-6 receptor bind to and capture IL-6 receptor or IL-6,respectively, so as to inhibit specifically the binding of IL-6 to IL-6receptor. As a result, they do not allow propagating of the biologicalactivity of IL-6, and thereby block signal transduction by IL-6.

Partial peptides of IL-6 or partial peptides of IL-6 receptor arepeptides are peptides comprising part or all of the amino acid sequenceinvolved in the binding of IL-6 and IL-6 receptor in the amino acidsequences of IL-6 or IL-6 receptor. Such peptides comprise usually 10-80amino acid residues, preferably 20-50 amino acid residues, and morepreferably 20-40 amino acid residues.

Partial peptides of IL-6 or partial peptides of IL-6 receptor specifythe regions involved in the binding of IL-6 and IL-6 receptor in theamino acid sequence of IL-6 or IL-6 receptor, and part or all of theamino acid sequence can be prepared by a commonly known method such asgene engineering technology or peptide synthesis.

In order to prepare partial peptides of IL-6 or partial peptides of IL-6receptor by gene engineering technology, a DNA sequence encoding thedesired peptide can be integrated into an expression vector so that theymay be obtained according to the above-mentioned methods for expression,production, and purification of recombinant antibody.

In order to prepare partial peptides of IL-6 or partial peptides of IL-6receptor by peptide synthesis, a commonly used method in peptidesynthesis such as solid-phase synthesis or liquid-phase synthesis can beused.

Specifically, methods described in “Zoku Iyakuhinno Kaihatsu, Vol. 14:Peptide Synthesis” edited by Haruaki Yajima, Hirokawa Shoten, 1991, canbe used. As the solid-phase synthesis, there can be used a method inwhich an amino acid corresponding to the C-terminal of the peptide to besynthesized is bound to a support insoluble in organic solvents, andthen a reaction in which amino acids of which α-amino group and a sidechain functional group has been protected with a suitable protectinggroup is condensed one by one in the direction of from the C-terminal tothe N-terminal and a reaction in which said protecting group of theα-amino group of the amino acid or the peptide bound to the resin iseliminated therefrom are alternately repeated to extend the peptidechain. The solid-phase peptide synthesis is roughly divided in the Bocmethod and the Fmoc method depending on the type of protecting groupsused.

After thus synthesizing the peptide of interest, a deprotecting reactionor a cleavage reaction of the peptide chain from the support may beperformed. For the cleavage reaction of peptide chains, the Boc methodemploys hydrogen fluoride or trifluoromethanesulfonic acid, or the Fmocmethod usually employs TFA. In the Boc method, the above protectedpeptide resin is treated in the presence of anisole in hydrogenfluoride. Subsequently, the elimination of the protecting group and thecleavage from the support may be performed to collect the peptide.Lyophilization of this yields crude peptide. On the other hand, in theFmoc method, the deprotection reaction and the cleavage reaction of thepeptide chain from the support may be performed in a manner similar tothe one mentioned above.

The crude peptide obtained may be subjected to HPLC to separate andpurify it. In its elution, a water-acetonitrile solvent commonly used inprotein purification may be used under an optimal condition. Fractionscorresponding to the peaks of the chromatographic profile are harvestedand then lyophilized. For the peptide fractions thus purified, molecularweight analysis by mass spectroscopy, analysis of amino acidcomposition, or analysis of amino acid sequence is performed foridentification.

Specific examples of IL-6 partial peptides and IL-6 receptor partialpeptides have been disclosed in Japanese Unexamined Patent Publication(Kokai) No. 2-188600, Japanese Unexamined Patent Publication (Kokai) No.7-324097, Japanese Unexamined Patent Publication (Kokai) No. 8-311098,and U.S. Pat. Publication U.S. Pat. No. 5,210,075.

The inhibitory activity of IL-6 signal transduction by IL-6 antagonistof the present invention can be evaluated using a commonly known method.Specifically, IL-6-dependent human myeloma line (S6B45, KPMM2), humanLennert T lymphoma line KT3, or IL-6-dependent HN60.BSF2 cells arecultured, to which IL-6 is added, and at the same time, in the presenceof IL-6 antagonist, the incorporation of ³H labeled thymidine by theIL-6 dependent cells is determined. Alternatively, ¹²⁵I-labeled IL-6 andIL-6 antagonist, at the same time, are added, and then ¹²⁵I-labeled IL-6that bound to the IL-6-ecpressing cells is determined for evaluation. Inthe above assay system, in addition to the group in which the IL-6antagonist is present, a negative control group in which contains noIL-6 antagonist is set up, and the results obtained in both are comparedto evaluate the IL-6-inhibiting activity by IL-6 antagonist.

As shown in Examples below, as therapeutic effects was observed byadministration of anti-IL-6 receptor antibody to children suffering fromchronic arthritis, IL-6 antagonists such as anti-IL-6 receptor antibodywere shown to have a therapeutic effect for chronic arthritides ofchildhood-related diseases.

Subjects to be treated in the present invention are mammals. The subjectmammals to be treated are preferably humans.

Therapeutic agents for chronic arthritides of childhood-related diseasesof the present invention may be administered orally or parenterally andsystemically or locally. For example, intravenous injection such as dripinfusion, intramuscular injection, intraperitoneal injection,subcutaneous injection, suppositories, enema, oral enteric coatedtablets, and the like may be selected, and the dosage regimen may beselected as appropriate depending on the age and conditions of thepatient. The effective dose is chosen from the range of 0.01 mg to 100mg per kg of body weight per administration. Alternatively, the dosageof 1 to 1000 mg, preferably 5 to 50 mg per patient may be selected.

Preferable dose and method for administering is, for example in the caseof anti-IL6 receptor antibody, an effective dose that provides freeantibody in the blood, and specifically, 0.5 mg to 40 mg, and preferably1 mg to 20 mg per 1 kg body weight per month (four weeks), which isadministered at once, or divided to several parts and administered, forexample, twice/week, once/week, once/two weeks, once/four weeks, etc,for example intravenously for example by dripping, or subcutaneously.Administering schedule may be adjusted by elongating intervals fromtwice/week or once/week to once/two weeks, once/three weeks, once/fourweeks, etc, dependent on observation of symptoms, and blood testprofile.

Therapeutic agents for chronic arthritides of childhood-related diseasesof the present invention may contain pharmaceutically acceptablecarriers and additives depending on the route of administration.Examples of such carriers or additives include water, a pharmaceuticallyacceptable organic solvent, collagen, polyvinyl alcohol,polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulosesodium, polyacrylic sodium, sodium alginate, water-soluble dextran,carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose,xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, propyleneglycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol,stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose,pharmaceutically acceptable surfactants and the like. Actual additivesused are chosen from, but not limited to, the above or combinationsthereof depending on the dosage form.

EXAMPLES

The present invention will be explained more specifically below withreference to Working Examples and Reference Examples, but it is to benoted that the present invention is not limited by these examples in anyway.

Working Example 1

A patient (5 years old, male) with systemic-onset type juvenilerheumatoid arthritis having the following history was subjected to a MRA(humanized anti-IL-6 receptor antibody) treatment.

History Before the Treatment

Symptoms developed with relaxation heat (one-peak fever at about 40° C.for consecutive days), arthralgia at both knees, and anthema. Afterdiagnosing based on leukocytosis, negative anti-nuclear antibody,negative rheumatoid factor, increased erythrocyte sedimentation rate,high CRP levels, etc., an aspirin administration was started, but noimprovement in relaxation heat and arthralgia was observed and thegeneral condition was aggravated. Thus, it was changed to an oral bolusadministration of steroid (prednisolone 30 mg/day) to see an improvementin various symptoms. However, with the gradual decrease in prednisolone,symptoms recurred at 10 mg/day, the patient was rehospitalized,subjected to a methylprednisolone (mPSL) pulse therapy andplasmapheresis, and furthermore the combined use of cyclosporin A (Cs A)was carried out with no improvement. Symptoms were severe (leukocytecount 25400/μl, CRP 11.2 mg/dL), and a plasmapheresis+mPSL pulsetherapy+Cs A was carried out and the patient entered into remission. Asan aftertreatment, prednisolone+froben was given to control relaxationheat, and a decline in fever was clinically noted, butinflammation-related hematology tests remained at high values (CRP>5mg/dL), and after discharge from hospital, relaxation heat wasperiodically observed, but treatment and observation were continuedmainly on an ambulatory basis. However, the patient started to complainof backache that was aggravated by fever, and after close examinationusing MBI etc., destructive damages were noted in the fourth and thefifth thoracic vertebras suggesting that they are compression fracture.Due to the necessity of relief to the thoracic vertebras, bed rest wascontinued for about one year, and accordingly muscles of the lower limbshave markedly weakened which rendered walking completely impossible. Thetriadic therapy of prednisolone+froben+Cs A was continued but CRP neverdropped to 5 mg/dL or lower.

The Result of Treatment

Administration started at 2 mg/kg. Since no side effects were seen, thedosage was increased to 4 mg/kg in a once per week administration. Feverthat had been noted until then disappeared quickly, and about two weekslater CRP became negative. General malaise was cleared, and the patientsomewhat improved. It became possible to decrease prednisolone graduallyand has decreased to 1 mg/day.

From the above results, MRA was found to be effective for the treatmentof chronic arthritides diseases of childhood of which symptoms could notbe controlled even with nonsteroidal anti-inflammatory drugs such asaspirin and froben, long term bolus steroids (for example prednine andmedrol), and immunosuppressants such as cyclosporin A and methotrexate.Therefore, it can be said that IL-6 antagonist, in particular anti-IL-6receptor antibody, is effective as a therapeutic agent for chronicarthritides diseases of childhood, specifically the systemic onset typeof the ARA classification, the systemic onset type of the EULARclassification, the systemic onset type of the ILAR classification, andthe SPRASH syndrome of the present inventors' classification.

Working Example 2

A 22-year old female. In April 1998, erythema punctatum appeared at thefemur, the recordial region, and fingers, and in May, arthralgia at theshoulder, the elbow and the knee, and fever between 38 and 39° C.appeared. Though nonsteroidal anti-inflammatory drugs (NSAIDs) werestarted, fever persisted, and in July, with leukocyte count at 18100/μl,CRP at 18.3 mg/dl, and serum ferritin at 440 ng/ml, the patient wasdiagnosed as having adult Still's disease. From early January 2000,fever between 39 and 40° C. and arthralgia appeared, which were believedto be a flare-up of adult Still's disease (CRP 15.8 mg/dl, ferritin,205.8 ng/ml).

Since it was difficult to reduce the dosage of steroids, methotrexate(MTX) and cyclosporin A (Cs A) were used in combination, but this couldnot control the progress of the disease, which aggravated breathing, andthus the patient was placed under the control of artificial respiration.Though the disease was somewhat improved by a steroid therapy, atreatment with humanized anti-IL-6 receptor antibody (MRA) was startedbecause of the complication of severe osteoporosis. MRA (200 mg) wasintravenously drip-infused for every two weeks. The inflammatoryreaction became negative on day 6 after the administration, anddecreases in the amount of corticosteroids progressed smoothly, and nosevere side effects were observed.

From the above results, MRA was found to be effective for the treatmentof adult Still's disease of which symptoms could not be controlled evenwith the combined use of MTX and CsA. Therefore, it can be said thatIL-6 antagonist, in particular anti-IL-6 receptor antibody, is effectiveas a therapeutic agent for Still's disease, specifically adult Still'sdisease.

Reference Example 1 Preparation of Human Soluble IL-6 Receptor

Using a plasmid pBSF2R.236 containing cDNA that encodes IL-6 receptorobtained by the method of Yamasaki et al. (Yamasaki et al., Science(1988) 241, 825-828), soluble IL-6 receptor was prepared by the PCRmethod. The plasmid pBSF2R.236 was digested with a restriction enzymeSph I to obtain IL-6 receptor cDNA, which was inserted into mp18(manufactured by Amersham). Using a synthetic primer designed tointroduce a stop codon into IL-6 receptor cDNA, mutation was introducedinto IL-6 receptor cDNA by the PCR method in an in vitro mutagenesissystem (manufactured by Amersham). By this procedure, the stop codon wasintroduced at the position of amino acid 345, and cDNA encoding solubleIL-6 receptor was obtained.

In order to express soluble IL-6 receptor in CHO cells, it was ligatedto a plasmid pSV (manufactured by Pharmacia) to obtain a plasmidpSVL344. Soluble IL-6 receptor cDNA digested with HindIII-SalI wasinserted into a plasmid pECEdhfr containing the cDNA of dhfr to obtain aCHO cell-expressing plasmid pECEdhfr344.

Ten μg of plasmid pECEdhfr344 was transfected to a dhfr-CHO cell lineDXB-11 (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA (1980) 77,4216-4220) by the calcium phosphate precipitation method (Chen, C. etal., Mol. Cell. Biol. (1987) 7, 2745-2751). The transfected CHO cellswere cultured for three weeks in a nucleoside-free αMEM selection mediumcontaining 1 mM glutamine, 10% dialyzed FCS, 100 U/ml penicillin and 100μ/ml streptomycin.

The selected CHO cells were screened by the limiting dilution method toobtain a single CHO cells clone. The CHO cell clone was amplified with20 nM-200 nM of methotrexate to investigate a human soluble IL-6receptor-producing CHO cell line 5E27. The CHO cell line 5E27 wascultured in a Iscov modified Dulbecco medium (IMDM, manufactured byGibco) supplemented with 5% FBS. The culture supernatant was collectedand the concentration of soluble IL-6 receptor in the culturesupernatant was determined by ELISA. The result confirmed the presenceof soluble IL-6 receptor in the culture supernatant.

Reference Example 2 Preparation of Anti-Human IL-6 Antibody

Ten μg of tissue-type IL-6 (Hirano et al., Immunol. Lett. (1988) 17, 41)was used with Freund's complete adjuvant to immunize BALB/c mice, andthis was repeated every week until anti-IL-6 antibody can be detected inthe serum. Immune cells were removed from the local lymph nodes, andwere fused with a myeloma cell line P3U1 using polyethylene glycol 1500.Hybridomas were selected by the method of Oi et al. (Selective Methodsin Cellular Immunology, W.H. Freeman and Co., San Francisco, 351, 19080)using the HAT culture medium to establish a hybridoma producinganti-human IL-6 antibody.

The hybridoma producing anti-human IL-6 antibody was subjected to anIL-6 binding assay in the following manner. Thus, a 96-well microtiterplate (manufactured by Dynatech Laboratories, Inc., Alexandria, Va.)made of flexible polyvinyl was coated overnight with 100 μl of goatanti-mouse Ig (10 μl/ml, manufactured by Cooper Biomedical, Inc.,Malvern, Pa.) in 0.1 M carbonate hydrogen carbonate buffer (pH 9.6) at4° C. Then, the plate was treated in 100 μl of PBS containing 1% bovineserum albumin (BSA) at room temperature for 2 hours.

After the plate was washed in PBS, 100 μl of the hybridoma culturesupernatant was added to each well, and incubated overnight at 4° C.After washing the plate, ¹²⁵I-labelled recombinant type IL-6 was addedto each well to 2000 cpm/0.5 ng/well, and after washing, radioactivityof each well was measured by a gamma counter (Beckman Gamma 9000,Beckman Instruments, Fullerton, Calif.). Of 216 hybridoma clones, 32hybridoma clones were positive in the IL-6 binding assay. From amongthese clones, finally MH166.BSF2, a stable clone, was selected.Anti-IL-6 antibody MH166 has a subtype of IgG1 κ type.

Then, using a IL-6-dependent mouse hybridoma clone MH60.BSF2, aneutralizing activity with regard to the growth of the hybridoma byMH166 antibody was investigated. MH60.BSF2 cells were aliquoted to1×10⁴/200 μl/well, to which a sample containing MH166 antibody wasadded, and cultured for 48 hours. After adding 0.5 μCi/well of³H-thymidine (New England Nuclear, Boston, Mass.), culturing wascontinued for further six hours. The cells were placed on a glass filterpaper, and were treated by an automated harvester (Labo Mash ScienceCo., Tokyo, Japan). As the control, rabbit anti-IL-6 antibody was used.

As a result, MH166 antibody inhibited ³H-thymidine incorporation byMH60.BSF2 cells induced by IL-6 in a dose dependent manner. Thisrevealed that MH166 antibody neutralizes the activity of IL-6.

Reference Example 3 Preparation of Anti-Human IL-6 Receptor Antibody

Anti-IL-6 receptor antibody MT18 prepared by the method of Hirata et al.(Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906) was conjugated toa CNBr-activated Sepharose 4B (manufactured by Pharmacia Fine Chemicals,Piscataway, N.J.) to purify IL-6 receptor (Yamasaki et al., Science(1988) 241, 825-828). A human myeloma cell line U266 was solubilizedwith 1 mM p-paraaminophenylmethanesulfonyl fluoride hydrochloride(manufactured by Wako Pure Chemicals) (digitonin buffer) containing 1%digitonin (manufactured by Wako Pure Chemicals), 10 mM triethanolamine(pH 7.8), and 0.15 M NaCl, and was mixed with MT18 antibody conjugatedto Sepharose 4B beads. Subsequently, the beads were washed six times inthe digitonin buffer to prepare a partially purified IL-6 receptor.

BALB/c mice were immunized with the above partially purified IL-6receptor obtained from 3×10⁹ U266 cells four times every ten days, andthen a hybridoma was prepared according to a standard method. Theculture supernatant of the hybridoma from growth-positive wells wereexamined for the biding activity to IL-6 receptor in the followingmanner. 5×10⁷ U266 cells were labelled with ³⁵S-methionine (2.5 mCi),and were solubilized with the above digitonin buffer. The solubilizedU266 cells were mixed with 0.04 ml of MT18 antibody conjugated toSepharose 4B beads, and then washed for six times in the digitoninbuffer. Using 0.25 ml of the digitonin buffer (pH 3.4),³⁵S-methionine-labeled IL-6 receptor was eluted, which was neutralizedwith 0.025 ml of 1M Tris, pH 7.4.

0.05 ml of the hybridoma culture supernatant was mixed with 0.01 mlProtein G Sepharose (manufactured by Pharmacia). After washing, theSepharose was incubated with 0.005 ml solution of ³⁵S-labeled IL-6receptor solution. The immunoprecipitated substances were analyzed bySDS-PAGE to study the culture supernatant of hybridoma that reacts withIL-6 receptor. As a result, a reaction-positive hybridoma clone PM-1 wasestablished. Antibody produced from the hybridoma PM-1 had the IgG1 κsubtype.

The activity of the antibody produced by the hybridoma PM-1 to inhibitthe binding of IL-6 to IL-6 receptor was evaluated using a human myelomacell line U266. Human recombinant IL-6 was prepared from E. coli (Hiranoet al., Immunol. Lett. (1988) 17, 41-45), and was labeled with ¹²⁵Iusing the Bolton-Hunter reagent (New England Nclear, Boston, Mass.)(Taga et al., J. Exp. Med. (1987) 166, 967-981).

4×10⁵ U266 cells were cultured with a culture supernatant of 70%(v/v)hybridoma PM-1 and 14000 CPM of ¹²⁵I-labeled IL-6 for one hour. Seventymicroliters of a sample was layered onto 300 μl of FCS in a 400 μlmicrofuge polyethylene tube, centrifuged, and then the radioactivity ofthe cells were measured.

The result revealed that the antibody produced by the hybridoma PM-1inhibits the binding of IL-6 to IL-6 receptor.

Reference Example 4 Preparation of Anti-Mouse IL-6 Receptor Antibody

A monoclonal antibody against mouse IL-6 receptor was prepared by themethod of Saito, T. et al., J. Immunol. (1991) 147, 168-173.

CHO cells that produce soluble mouse IL-6 receptor were cultured in anIMDM culture medium supplemented with 10% FCS. From the culturesupernatant, soluble mouse IL-6 receptor was purified using an affinitycolumn in which anti-mouse IL-6 receptor antibody RS12 (see the aboveSaito, T. et al.) was immobilized to the Affigel 10 gel (manufactured byBiorad).

Fifty μg of soluble mouse IL-6 receptor thus obtained was mixed withFreund's complete adjuvant, which was intraperitoneally injected to theabdomen of Wistar rats. Two weeks later, the rats received boosterimmunization with Freund's incomplete adjuvant. On day 45, spleen cellswere removed from the rats, and 2×10⁸ of the cells were subjected tocell fusion with 1×10⁷ mouse myeloma cells P3U1 with 50% PEG1500(manufactured by Boehringer Mannheim) using a standard method, and thehybridoma were then screened with the HAT medium.

After adding the culture supernatant to a plate coated with rabbitanti-rat IgG antibody (manufactured by Cappel), soluble mouse IL-6receptor was reacted thereto. Then, using an ELISA method employingrabbit anti-mouse IL-6 receptor antibody and alkalinephosphatase-labelled sheep anti-rabbit IgG, hybridomas that produceantibodies against soluble mouse IL-6 receptor were screened. Thehybridoma clones for which antibody production was confirmed weresubjected to subscreening twice to obtain a single hybridoma clone. Thisclone was designated as MR16-1.

A neutralizing activity in signal transduction of mouse IL-6 by theantibody produced by this hybridoma was examined using ³H-thymidineincorporation that employs MH60.BSF2 cells (Matsuda, T. et al., J.Immunol. (1988) 18, 951-956). To a 96-well plate, MH60.BSF2 cells wereprepared to 1×10⁴ cells/200 μl/well. To this plate were added 10 μg/mlof mouse IL-6 and MR16-1 antibody or RS12 antibody at 12.3-1000 ng/ml,and cultured at 37° C. in 5% CO₂ for 44 hours, followed by the additionof 1 μCi/well of ³H-thymidine. Four hours later, the incorporation of³H-thymidine was measured. As a result, MR16-1 antibody inhibited the³H-thymidine incorporation by MH60.BSF2 cells.

Thus, it was revealed that antibody produced by the hybridoma MR16-1(FERM BP-5874) inhibits the binding of IL-6 to IL-6 receptor.

1-15. (canceled)
 16. A method for treatment of adult-onset Sill'sdisease, comprising administering a subcutaneous injection of anantibody against human IL-6 receptor to a human patient who needs saidtreatment, wherein the antibody against the IL-6 receptor is an antibodywhich inhibits binding of IL-6 to the IL-6 receptor by binding to theIL-6 receptor.
 17. The method of claim 16, wherein the antibody againstIL-6 receptor is a monoclonal antibody.
 18. The method of claim 16,wherein the antibody against L-6 receptor is recombinant.
 19. The methodof claim 17, wherein the monoclonal antibody against IL-6 receptor isthe PM-1 antibody (FERM BP-2998) or a humanized or chimeric versionthereof.
 20. The method of claim 16, wherein treatment further comprisesadministration of methotrexate, non-steroidal anti-inflammatory drugs(NSAIDs), or corticosteroids.
 21. A method for treatment ofsystemic-onset type juvenile rheumatoid arthritis, comprisingadministering an intravenous injection of an antibody against human IL-6receptor to a human patient who needs said treatment, wherein theantibody against the IL-6 receptor is an antibody which inhibits bindingof IL-6 to the IL-6 receptor by binding to the IL-6 receptor, whereinthe antibody against human IL-6 receptor is administered in a dose of1-20 mg/kg body weight.
 22. The method of claim 21, wherein the antibodyagainst human IL-6 receptor is administered in a dose of 8-12 mg/kg bodyweight.
 23. The method of claim 21, wherein the antibody against humanIL-6 receptor is administered once every two weeks.
 24. The method ofclaim 21, wherein the antibody against IL-6 receptor is a monoclonalantibody.
 25. The method of claim 21, wherein the antibody against IL-6receptor is recombinant.
 26. The method of claim 24, wherein themonoclonal antibody against IL-6 receptor is the PM-1 antibody (FERMBP-2998) or a humanized or chimeric version thereof.
 27. The method ofclaim 21, wherein treatment further comprises administration of one ormore of methotrexate, a non-steroidal anti-inflammatory drug (NSAID), ora corticosteroid.